The factors that affect the performance of chromatographic columns include:1、 Physical factors:
(1) Purity of silica gel: The purity of silica gel and the concentration of residual metal ions. Impurities in silica gel can affect the peak shape of compounds, and high metal content on the surface of silica gel can affect the peak shape of alkaline compounds, leading to tailing.
(2) The size of the chromatographic column and the length and inner diameter of the packed bed. Increasing the length of the chromatographic column can improve column efficiency to a certain extent, but it can also increase pressure and lead to peak broadening; Wide column diameter can increase sample loading capacity, but it also increases lateral diffusion, which can lead to peak broadening. Narrow column diameter can save solvents and reduce lateral diffusion, but it requires high pressure and high system requirements.
(3) Spherical particles have high column efficiency, good reproducibility, uniform column bed structure, irregular column bed structure, uneven flow phase line velocity, and are prone to spectral band broadening; The average particle diameter, the smaller the particle size, the higher the column efficiency, and the higher the column pressure. The wider the particle size distribution, the lower the column efficiency and the higher the pressure.
(4) Particle surface area: the sum of the outer surface and inner pore surface of the particle, expressed in m2/g. Relatively speaking, high surface area has stronger retention capacity, larger column capacity, and higher separation efficiency for the sample. Low surface area can reach equilibrium faster and is more suitable for gradient elution programs.
(5) Aperture: The average size of a particle's pores or cavities, ranging from 80 to 300? Large pore size filler particles can prolong the residence time of large molecular solutes on the surface of the filler, improve separation, so large pore size fillers are suitable for separating large molecular compounds or molecules with larger hydrodynamic volumes.
2、 Chemical factors:
(1) Bonding type and bonding phase: The bonding phase molecules are connected to the substrate at a single point through monomer bonding, which can improve mass transfer rate and shorten column equilibrium time,; Polymer bonding forms a multi-point connection between the bonded phase molecules and the matrix, which can increase the stability of the chromatographic column and increase the sample loading capacity. Different bonding phases directly affect the retention behavior of compounds.
(2) Carbon coverage: High carbon coverage can improve resolution and reproducibility, but the analysis time is long.
(3) End capping: There are some residual silicon hydroxyl groups in the silica gel bonded phase filler that are not capped, as shown in the figure. End capping can reduce the reaction between the tested component and the acidic silicon hydroxyl groups remaining on the surface of the silica gel, improve retention and peak behavior, which is particularly important for alkaline compounds. And different capping techniques can also directly affect the efficiency of the chromatographic column.